1. Field of the Invention
The invention relates to the therapeutic use of oligonucleotides, both in the antisense approach, and as immunostimulatory agents.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al.,Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Antl. Acad. Sci. USA 85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
More recently, several researchers have demonstrated the validity of the antisense approach to therapeutic treatment of disease. Crooke, Antisense Nucleic Acid Drug Dev. 8: vii-viii, discloses the successful marketing approval of a phosphorothioate oligonucleotide for the treatment of human cytomegalovirus-induced retinitis. Unfortunately, the use of phosphorothioate oligonucleotides has become more complex than originally expected. Certain effects caused by phosphorothioate oligonucleotides could not be explained by the expected antisense mechanism. For example, McIntyre et al., Antisense Res. Dev. 3: 309-322 (1993) teaches that a xe2x80x9csensexe2x80x9d phosphorothioate oligonucleotide causes specific immune stimulation. This and other side effects have complicated the picture for phosphorothioate oligonucleotides. Zhaoet al., Biochemical Pharmacology 51:173-182 (1996) discloses immune stimulation mediated by two CpG-containing oligonucleotides, one complementary to the gag gene from HIV-1 (5xe2x80x2-CTCTCGCACCCATCTCTCTCCTTCT-3xe2x80x2), and the other complementary to the rev gene of HIV-1 (5xe2x80x2-TCGTCGCTGTCTCCGCTTCTTCTTGCC-3xe2x80x2).
On the other hand, the observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in developing this side effect as a therapeutic tool. These efforts have focussed on phosphorothioate oligonucleotides containing the dinucleotide CpG. Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity. Krieg et al., Nature 371: 546-549 (1995) discloses that phosphorothioate CpG-containing oligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest. 98: 1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu et al., Vaccine 16: 1216-124 (1998) teaches that CpG-containing phosphorothioate oligonucleotides enhance immune response against influenza virus. McCluskie and Davis, The Journal of Immunology 161: 4463-4466 (1998) teaches that CpG-containing oligonucleotides act as potent immunostimulatory agents, enhancing immune response against hepatitis B surface antigen. Agrawal, U.S. Pat. No. 5,968,909, teaches that backbone modification of C or G suppresses this effect.
These reports make clear that there is a need to be able to modulate the immune response caused by CpG-containing oligonucleotides. Ideally, such modulation should include decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications.
The invention provides methods for modulating the immune response caused by CpG-containing oligonucleotides. The methods according to the invention enable both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications. Thus, the invention further provides oligonucleotides having optimal levels of immunostimulatory effect for either application and methods for using such oligonucleotides.
The present inventor has surprisingly discovered that positional modification of CpG-containing oligonucleotides dramatically affects their immunostimulatory capabilities. In particular, 2xe2x80x2 alkylation or alkoxylation of oligonucleotides at particular positions 5xe2x80x2 or 3xe2x80x2 to the CpG dinucleotide either enhances or reduces their immunostimulatory effect.
In a first aspect, the invention provides a method for reducing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing a 2xe2x80x2 substituted nucleoside into the oligonucleotide at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide, wherein at least one nucleoside is not a 2xe2x80x2-O-methylribonucleoside and the oligonucleotide is not complementary to the gag or rev gene of human immunodeficiency virus type 1.
In a second aspect, the invention provides a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 2xe2x80x2 substituted nucleoside at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide, wherein at least one nucleoside is not a 2xe2x80x2-O-methylribonucleoside and the oligonucleotide is not complementary to the gag or rev gene of human immunodeficiency virus type 1.
In a third aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, the method comprising administering to the mammal a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 2xe2x80x2 substituted nucleoside at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide, wherein at least one nucleoside is not a 2xe2x80x2-O-methylribonucleoside and the oligonucleotide is not complementary to the gag or rev gene of human immunodeficiency virus type 1.
In a fourth aspect, the invention provides a method for increasing the immunostimulatory effect of a CpG-containing oligonucleotide, wherein the oligonucleotide is not an antisense oligonucleotide complementary to Ha-ras or the gag or rev gene of human immunodeficiency virus type 1. The method according to this aspect of the invention comprises introducing into the oligonucleotide a 2xe2x80x2 substituted nucleoside at a position selected from the group consisting of second nucleoside 5xe2x80x2 to the CpG dinucleotide, third nucleoside 5xe2x80x2 to the CpG dinucleotide, fourth nucleoside 5xe2x80x2 to the CpG dinucleotide, fifth nucleoside 5xe2x80x2 to the CpG dinucleotide, sixth nucleoside 5xe2x80x2 to the CpG dinucleotide, first nucleoside 3xe2x80x2 to the CpG dinucleotide, second nucleoside 3xe2x80x2 to the CpG dinucleotide, third nucleoside 3xe2x80x2 to the CpG dinucleotide, fourth nucleoside 3xe2x80x2 to the CpG dinucleotide, fifth nucleoside 3xe2x80x2 to the CpG dinucleotide, sixth nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide. In alternative embodiments the oligonucleotide may be an antisense oligonucleotide.
In a fifth aspect, the invention provides CpG-containing oligonucleotides having increased immunostimulatory effects, the oligonucleotide comprising a 2xe2x80x2 substituted nucleoside at a position selected from the group consisting of second nucleoside 5xe2x80x2 to the CpG dinucleotide, third nucleoside 5xe2x80x2 to the CpG dinucleotide, fourth nucleoside 5xe2x80x2 to the CpG dinucleotide, fifth nucleoside 5xe2x80x2 to the CpG dinucleotide, sixth nucleoside 5xe2x80x2 to the CpG dinucleotide, first nucleoside 3xe2x80x2 to the CpG dinucleotide, second nucleoside 3xe2x80x2 to the CpG dinucleotide, third nucleoside 3xe2x80x2 to the CpG dinucleotide, fourth nucleoside 3xe2x80x2 to the CpG dinucleotide, fifth nucleoside 3xe2x80x2 to the CpG dinucleotide, sixth nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof, wherein the oligonucleotide is not an antisense oligonucleotide complementary to Ha-ras or the gag or rev gene of human immunodeficiency virus type 1. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide.
In a sixth aspect, the invention provides a method for inducing an immune response in a mammal, the method comprising administering to the mammal an oligonucleotide comprising a 2xe2x80x2 substituted nucleoside at a position selected from the group consisting of second nucleoside 5xe2x80x2 to the CpG dinucleotide, third nucleoside 5xe2x80x2 to the CpG dinucleotide, fourth nucleoside 5xe2x80x2 to the CpG dinucleotide, fifth nucleoside 5xe2x80x2 to the CpG dinucleotide, sixth nucleoside 5xe2x80x2 to the CpG dinucleotide, first nucleoside 3xe2x80x2 to the CpG dinucleotide, second nucleoside 3xe2x80x2 to the CpG dinucleotide, third nucleoside 3xe2x80x2 to the CpG dinucleotide, fourth nucleoside 3xe2x80x2 to the CpG dinucleotide, fifth nucleoside 3xe2x80x2 to the CpG dinucleotide, sixth nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof, wherein the oligonucleotide is not an antisense oligonucleotide complementary to Ha-ras or the rev or gag gene of human immunodeficiency virus type 1. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide.